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Nov 19th 2012 Lecture 7: 1.Advanced Separations Methods: HPLC vs. UPLC vs. HILIC 2. Nanoflow vs. ESI 3. Applications; 4.Laser capture miscrodissection (LCM) Adriana Bora, PhD ME 330.80: Role of Chromatography & Mass 1 Spectrometry in Biological Research http://www.hopkinsmedicine.org/mams/ Technologies for Proteomics ME 330.80: Role of Chromatography & Mass 2 Spectrometry in Biological Research http://www.hopkinsmedicine.org/mams/ Nature biotechnology 28, 695-709, (2010) “omics” Workflow Sample Sample preparation Tissue Blood CSF Plasma Urine Serum Peptide/Protein Extraction, Desalting, Abundant Protein Depletion, Detergent Removal, etc. Separation HPLC/UPLC, HILIC, SEC, IEC, etc. Mass Spectrometry LC-MALDI, MALDI-TOF, QTOF, IT, Orbitrap, etc. Data Analysis MASCOT, SEQUEST, PROTEIN DISCOVERER, etc. ME 330.80: Role of Chromatography & Mass 3 Spectrometry in Biological Research http://www.hopkinsmedicine.org/mams/ Liquid Chromatography • Defined as separation of components of a mixture based upon the rates at which they elute from a stationary phase typically over a mobile phase gradient. • Ion exchange chromatography • Size exclusion chromatography • Adsorption chromatography ME 330.80: Role of Chromatography & Mass 4 Spectrometry in Biological Research http://www.hopkinsmedicine.org/mams/ Different Phases • Normal Phase – This is where the stationary bed is strongly polar (silica gel) and the mobile phase is largely non-polar such as hexane. • Reverse Phase – The stationary phase is nonpolar and the mobile phase are polar liquids such as methanol, acetonitrile, or water. The more non-polar substances have longer retention. Reference 1 ME 330.80: Role of Chromatography & Mass 5 Spectrometry in Biological Research http://www.hopkinsmedicine.org/mams/ Elution Types • Isocratic – where the eluent is at a fixed concentration. • Gradient – where the eluent concentration and strength are changing. Reference 1 ME 330.80: Role of Chromatography & Mass 6 Spectrometry in Biological Research http://www.hopkinsmedicine.org/mams/ Types of Liquid Chromatography (TLC) Paper Gravity Chrom. Chrom. Tsvett, 1903 Flash Chrom. 1978 HPLC 1952 UPLC 2004 ME 330.80: Role of Chromatography & Mass 7 Spectrometry in Biological Research http://www.hopkinsmedicine.org/mams/ HPLC Characteristics • Columns have small internal diameters (2-10 mm) usually made with a reusable material like stainless steel • High inlet pressures of several thousand psi’s and controlled flow of mobile phase • Precise sample introduction and small sample requirements • Special continual flow detectors that use small flow rates and low detection limits • Some are equipped with automated sampling devices • Rapid analysis with high resolution Reference 3 ME 330.80: Role of Chromatography & Mass 8 Spectrometry in Biological Research http://www.hopkinsmedicine.org/mams/ Stationary Phase in HPLC • Particle size 3 to 10 µm packed tightly with a pore size of 70 to 300 Å • Surface area of 50 to 250 m2/g • Bond phase density – number of adsorption sites per surface unit (1 to 5 per 1 nm). • Typical surface coatings: Normal phase (-Si-OH, -NH2) Reverse phase (C8, C18, Phenyl) Anion exchange (-NH4+) Cation exchange (-COO-) Reference 3 ME 330.80: Role of Chromatography & Mass 9 Spectrometry in Biological Research http://www.hopkinsmedicine.org/mams/ Mobile Phase in HPLC • • • • • • Purity of the solvents Detector compatibility Solubility of the sample Low viscosity Chemical inertness Reasonable price Reference 3 ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 10 http://www.hopkinsmedicine.org/mams/ Path of Mobile Phase Mobile Phase degassing HPLC Column Mobile Phase reservoir Mobile Phase mixing Rotary Sample Loop injector HPLC Pump HPLC Detector ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 11 http://www.hopkinsmedicine.org/mams/ How does it work HPLC/UPLC Technology? Chromatogram HPLC/UPLC column A Pumps B Detector Line Syringe wash wash Solvent proportioning Flow valve and monitor valve Secondary Detector: UV/Vis MS NMR Autosampler injector Waste Solvent A 5% Aq, ACN +0.1% FA Solvent B 95% Aq, ACN +0.1% FA Phytochem. Analysis, 2010, 21, 33-47, Allwood &Goodacre ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 12 http://www.hopkinsmedicine.org/mams/ HPLC Columns • HPLC Columns come in various sizes and many factors involving your analyte or the function of the column should be considered when selecting the appropriate one. • Some common dimensions: 10, 15, and 25 cm in length; • 3, 5, or 10 mm diameters; • 4 to 4.6um internal diameters Reference 3 ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 13 http://www.hopkinsmedicine.org/mams/ HPLC Detectors • Most HPLC instruments are equipped with optical detectors. • Light passes through a transparent low volume “flow cell” where the variation in light by UV Absorption, fluorescent emission, or change in refractive index are monitored and integrated to display Retention Time and Peak Area. • Typical flow rates are 1 mL/min. and a flow cell volume of 5-50 µL. Reference 3 ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 14 http://www.hopkinsmedicine.org/mams/ Common HPLC Detectors • Refractive Index (RI) - universal • Evaporative Light Scattering Detector (ELSD) – universal • UV/VIS light – selective • Fluorescence – selective • Electrochemical (ECD) selective • Mass Spec (MS) - universal Reference 3 ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 15 http://www.hopkinsmedicine.org/mams/ Mass Spectrometer • Thermospray – mobile phase is directed to a capillary column that is heated and points at a skimmer cone. (Too much build up on orifice) • Electrospray (ESI) – analytes are charged upon exiting the capillary tube and cross sprayed with nitrogen. The charge particles cause a “Coulomb explosion” making smaller droplets of analyte to enter the skimmer cone. • Atmospheric Pressure Chemical Ionization (APCI) – Analyte is heated by a ceramic tip on the column, cross flow of nitrogen decreases the droplet size, and a “corona discharge” charges the particles to enter the detector. Reference 3 ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 16 http://www.hopkinsmedicine.org/mams/ Why HPLC? • HPLC works with compounds of higher molecular weights and polarity. • Many biological samples are charged such as DNA and proteins. • HPLC can be used with larger sample sizes and sample recovery to continue synthesis • Good at separating stereoisomers; techniques that employ heat (GC) can cause racemization during analysis. Reference 3 ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 17 http://www.hopkinsmedicine.org/mams/ UltraPerformance Liquid Chromatography (UPLC ) Technology • In 2004, further advances in instrumentation and column technology were made to achieve very significant increase in: RESOLUTION SPEED SENSITIVITY Increase separation EFFICIENCY • Columns with smaller particles [<1.7um] • Mobile phase delivery is done at >15,000psi ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 18 http://www.hopkinsmedicine.org/mams/ Contrasting HPLC and UPLC • UPLC gives faster results with better resolution • UPLC uses less of valuable solvents like acetonitrile which lowers cost • The reduction of solvent use is more environmentally friendly Reference 6 ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 19 http://www.hopkinsmedicine.org/mams/ UPLC columns an ethylene bridged hybrid (BEH) structure • Superior mechanical strength • Efficiency • High pH stability and peak shape for bases • C8; C18;Phenyl; HILIC • pH range 1-12 • Max pressure 15,000psi • Particle size 1.7um • Pore diameter/volume 130A 0.7 mL/g • Surface Area 185 m^2/g • • • • Peptides Proteins Oligonucleotides DNA/RNA Amino acids Plate height UPLC principle The evolution of particle sizes over the last three decades. ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 21 http://www.hopkinsmedicine.org/mams/ Why is UPLC more efficient • Peak capacity (P) is the number of peaks that can be resolved in a specific amount of time. • P is proportional to the inverse of the square root of the Number of theoretical plates (N): N = L/H • Lower plate heights generate a smaller number of plates • Plate heights are correlated through the Van Deemter equation ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 22 http://www.hopkinsmedicine.org/mams/ Why we need UPLC Technology? • Metabolomics is the comprehensive assessment of endogenous metabolites of low-molecular weight (<1,000 Da)of a biological system. • These small molecules, including peptides , amino acids , nucleic acids , carbohydrates , organic acids, vitamins , polyphenols , alkaloids and inorganic species act as small-molecule biomarkers that represent the functional phenotype in a cell , tissue or organism. • Applications: drug discovery, toxicology, nutrition, cancer, natural product discovery, etc. • These large-scale analyses of metabolites are intimately bound to advancements in ultra-performance liquid chromatography–electrospray (UPLC) technologies and have emerged in parallel with the development of novel mass analyzers and hyphenated techniques. ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 23 http://www.hopkinsmedicine.org/mams/ Chromatograms of simvastatin • hypolipidemic drug • control elevated cholesterol ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 24 http://www.hopkinsmedicine.org/mams/ Chromatogram showing separation of Telmisartan and its degradation products in a mixture of stressed samples Angiotensin II receptor antagonist a) UPLC used to control hypertension b) HPLC Reference 3 ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 26 http://www.hopkinsmedicine.org/mams/ Hydrophilic Interaction Liquid Chromatography (HILIC) • The principle of HILIC was described by Samuelson and Sjostrom (1952) for the separation of monosaccharides using anion exchange rasin as stationary phase; Samuelson O, Sjöström E. 1952. Utilization of Ion Exchangers in Analytical Chemistry. XXIV. Isolation of Monosaccharides. Sven Kem Tidskr64: 305–314. • The mechanism of retention is based on the hydrophilic partitioning of the analytes into the water-enriched stationary phase, and weak electrostatic interactions with either the positive or negative charge of the functional group. • HILIC separates polar molecules. • A sulfoalkylbetaine zwitterionic stationary phase. Cubbon et al, Jun 2009, Mass Spec. Reviews; Metabolomic applications of HILIC–LC–MS Reference 3 ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 27 http://www.hopkinsmedicine.org/mams/ HILIC application • Like the SCX method mentioned above, HILIC chromatography can be used to enrich PTM-modified sub-populations from complex biological samples. • Specifically, HILIC has been shown to enrich for glycosylation, N-acetylation, and phosphorylation. Peptides with N-acetyl modifications will elute as an early subfraction during a HILIC separation at relatively low water/organic mobile phase conditions. Phosphopeptides will elute within the middle of a HILIC separation, but with little contamination from non-phosphorylated species. Glycosylated peptides will be retained on the HILIC column while most sample components elute off; once high water/organic mobile phase ratios are reached, the remaining glycosylated peptides will elute as a purified sub-proteome. • HILIC offer a good alternative to RP chromatography for the analysis of highly polar metabolites such as carbohydrates, their phosphorylated derivatives, and glycolytic intermediates, which are poorly retained on RPs. Reference 3 ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 28 http://www.hopkinsmedicine.org/mams/ HILIC applications Reference 3 ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 29 http://www.hopkinsmedicine.org/mams/ In real life we mostly use orthogonal separations 2000 Nucleic proteins Gauci et al, J. Proteome Res. , 2009, 8(7), pp 3451-3463 NanoLC- ESI –MS • NanoLC (nLC) is named after the low flow rate (200-300 nL/min). • This uses very low sample volumes and (1µL) very high selectivity and sensitivity are possible. • nLC-ESI-IT-MS/MS is mostly used for the identification of proteins from very complex mixtures. ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 31 http://www.hopkinsmedicine.org/mams/ Gel Eluted Liquid Fraction Entrapment Electrophoresis (GELFrEE) - SDS PAGE Column + Cathode Chamber J.C. Tran; A.A. Doucette, Anal. Chem. 2008, 80, 1568-1573 Courtesy of John Tran Anode Chamber Collection Chamber Membrane Trap ME 330.80: Role of Chromatography &32 Mass Spectrometry in Biological Research 32 32 http://www.hopkinsmedicine.org/mams/ Top Down Proteomics Front-end separation Mass spectral data acquisition Protein identification by database search Intact protein separation based on molecular weight: Gel Eluted Liquid Fraction Entrapment Electrophoresis (GELFrEE) – Cathode + Anode SDS-PAGE Column MW Courtesy of Neil Kelleher Collection Tran, J. C.; Doucette, A. A., Anal. Chem. 2008, 1568-1573Chamber Membrane Trap Time (60-90 min) ME 330.80: Role of Chromatography &33 Mass Spectrometry in Biological Research 33 33 http://www.hopkinsmedicine.org/mams/ Gel Eluted Liquid Fraction Entrapment Electrophoresis (GELFrEE) Courtesy of John Tran Tran, J.C.; Doucette, A.A., Anal. Chem. 2008, 80, 1568-1573 ME 330.80: Role of Chromatography &34 Mass Spectrometry in Biological Research 34 34 http://www.hopkinsmedicine.org/mams/ GELFrEE Separation of Bacterial Proteome Mol Wt (kDa) 1 cm Column 212 100 15 15.5 16 17 18 20 collection time (min) 22 24 26 28 30 J.C. Tran; A.A. Doucette, Anal. Chem. 2008, 80, 1568-1573 35 40 45 60 75 90 std 54 39 30 20 7.3 Mol Wt (kDa) •1cm column gives faster and better separation over a broad mass range 3 cm Column 212 100 25 27 30 33 36 collection time (min) 42 48 54 60 66 72 87 102 112 157 202 std 54 39 30 20 7.3 ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 35 35 http://www.hopkinsmedicine.org/mams/ GELFrEE Recoveries J.C. Tran; A.A. Doucette, Anal. Chem. 2008, 80, 1568-1573 BSA Cytochrome C Ubiquitin 40 ng Loading ME 330.80: Role of Chromatography &36 Mass Spectrometry in Biological Research 36 36 http://www.hopkinsmedicine.org/mams/ Multiplex GELFrEE power terminals Collect Load Courtesy of John Tran plate - gasket gasket plate + gel columns cathode chamber plate J.C. Tran; A.A. Doucette, Anal. Chem. 2009, 81, 6201-6209 plate bolts collection anode chamber dialysis chamber membrane ME 330.80: Role of Chromatography &37 Mass Spectrometry in Biological Research 37 37 http://www.hopkinsmedicine.org/mams/ Multiplex GELFrEE Load Collect ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 38 38 http://www.hopkinsmedicine.org/mams/ Increased Loading Capacity J.C. Tran; A.A. Doucette, Anal. Chem. 2009, 81, 6201-6209 Overloaded Column kDa 250 150 100 75 50 37 16 17 18 19 20 collection time (min) 21 22 24 26 28 30 35 40 45 60 75 90 std 800 µg /column 0.4mm id 25 20 15 10 Pooled Fractions from Equivalent Overloaded Amount kDa 250 150 100 75 50 37 16 17 18 19 20 22 24 26 28 30 35 40 45 60 75 90 std 0.4mm id 800µg loaded in 8 different channels :100 mg / column 25 15 10 ME 330.80: Role of Chromatography & Mass 39 Spectrometry in Biological Research 39 39 http://www.hopkinsmedicine.org/mams/ Increased Throughput Fractions 1-16 collection time (min) kDa 250 150 100 75 50 37 25 16 17 18 19 20 22 24 26 28 30 35 40 45 60 75 90 std Yeast 15 10 kDa 250 150 100 75 50 37 25 20 Urine 15 10 kDa 250 150 100 75 50 37 25 20 15 10 Bacteria J.C. Tran; A.A. Doucette, Anal. Chem. 2009, 81, 6201-6209 ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 40 40 http://www.hopkinsmedicine.org/mams/ High reproducibility of different in channels S.cerevisiae Proteome J.C. Tran; A.A. Doucette, Anal. Chem. 2009, 81, 6201-6209 800ug per column 100ug per column ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 41 41 http://www.hopkinsmedicine.org/mams/ Identified proteins with LC MSMS S.cerevisiae Proteome •1120 proteins •428 unique proteins •1% false positive rate ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 42 42 http://www.hopkinsmedicine.org/mams/ Bottom Up Proteomics • Analyzing CSF using GelFrEE –LC-MS/MS 400 Adriana Bora; Carol Anderson; Muznabanu Bachani; Avindra Nath; Robert J. Cotter; J. Proteome Res. 2012, 11, 3143-3149. ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 43 43 http://www.hopkinsmedicine.org/mams/ Unseparated human CSF proteome Separated human CSF proteome with GelFrEE A) Silver stained images showing GelfrEE fractions for visualization of the prote separation found in each fraction. B) and C) show the reproducibility of the separation technique. Adriana Bora; Carol Anderson; Muznabanu Bachani; Avindra Nath; Robert J. Cotter; J. Proteome Res. 2012, 11, 3143-3149. ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 44 44 http://www.hopkinsmedicine.org/mams/ Sample Preparation Affinity Column 4 min Tryptic Digestion OnColumn Desalting C18 Reverse Phase HPLC SRM-MS Detection • Reduces variability to under 10% • Cutting sample prep time down to 10min • Reducing operating cost by 50% ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 45 45 http://www.hopkinsmedicine.org/mams/ Perfinity – Shimadzu 8030 triple quadrupole MS ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 46 46 http://www.hopkinsmedicine.org/mams/ Insulin protein digested on trypsin column using Perfinity System uV 550000 500000 450000 400000 Undigested protein 350000 300000 1min 250000 2min 200000 150000 4min 100000 6min 50000 0 8min -50000 -100000 -150000 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 min ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 47 47 http://www.hopkinsmedicine.org/mams/ Separation of CSF proteins using the Perfinity System CSF 30min gradient- 6min digestion-60 %B -40C mAU % 70 90 65 80 60 55 70 50 60 45 50 40 40 35 30 30 25 20 20 10 15 0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0 28.0 29.0 30.0 31.0 min ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 48 48 http://www.hopkinsmedicine.org/mams/ Laser capture miscrodissection (LCM) ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 49 http://www.hopkinsmedicine.org/mams/ ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 50 http://www.hopkinsmedicine.org/mams/ Applications of LCM ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 51 http://www.hopkinsmedicine.org/mams/ Cont… ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 52 http://www.hopkinsmedicine.org/mams/ Cont…. ME 330.80: Role of Chromatography & Mass Spectrometry in Biological Research 53 http://www.hopkinsmedicine.org/mams/